JPH0776570A - Isolation of abscisic acid - Google Patents

Abstract

PURPOSE:To carry out efficient isolation of abscisic acid in high selectivity and yield from a dilute solution of abscisic acid without concentration of the organic layer extracting abscisic acid using a reduced amount of the solvent. CONSTITUTION:The isolation process for abscisic acid comprises the extraction step of bringing the aqueous abscisic acid into contact with an organic solvent immiscible with water to transfer the abscisic acid into the organic solvent, the adsorption step of bringing the organic layer containing abscisic acid into contact with an adsorbent to transfer the abscisic acid into the adsorbent, the recycling step of recycling the organic layer from which abscisic acid is desorbed to the extraction step, and the isolation step of isolating abscisic acid from the solution separated. The isolated abscisic acid lower the purification load thereafter to give ABA of high purity inexpensively in an industrial scale.

Description

Detailed Description of the Invention

[0001]

The present invention relates to abscisic acid (hereinafter referred to as A
Abbreviated as BA).

ABA is an important compound as a natural plant hormone and is expected to have a wide range of uses.

[0003]

2. Description of the Related Art Conventionally, ABA produced by fermentation has been produced by liquid culture and solid culture of Sercospora rosicola and Botrytis cinerea. The obtained culture solution and culture product are extracted with an organic solvent and the extract solution is concentrated. Method (Japanese Patent Publication No. 60-6629, Japanese Patent Publication No. 61-35838), or a method in which the obtained culture solution is passed through an ion exchange resin and an adsorption resin and then eluted with a solvent to concentrate the eluate ( Japanese Patent Publication No. 3-6139) and the like are known.

[0004]

However, the former method is complicated because a large amount of solvent is used and the large amount of extract solution must be concentrated. Also. The latter method is complicated because it uses a large amount of resin and a large amount of elution solvent and concentrates a large amount of the eluent, and neither method is suitable for industrial production of ABA.

[0005]

Therefore, the present inventors have
The present invention has been achieved as a result of extensive studies on a method for efficiently and selectively isolating ABA from an aqueous solution containing ABA in a high yield.

That is, the present invention provides (i) an extraction step in which an aqueous solution containing ABA is contacted with an organic solvent immiscible with water to transfer the ABA from the aqueous solution to the organic solvent, (ii) the extraction in (i) The ABA-containing organic layer separated in the step is brought into contact with an adsorbent, and the ABA-containing organic layer is removed from the organic layer.
An adsorption step of adsorbing BA to the adsorbent, (iii)
(ii) the step of recycling the remaining organic layer on which the ABA is adsorbed in the adsorption step to (i) the extraction step, and (iv) the above
(ii) AB comprising the step of isolating the ABA from the adsorbent that has adsorbed the ABA in the adsorption step
It is a method of isolating A.

The present invention will be described in detail below.

In the present invention, first, (i) an aqueous solution containing ABA is brought into contact with an organic solvent immiscible with water and subjected to an extraction step of transferring the ABA from the aqueous solution to the organic solvent.

The aqueous solution containing ABA used in the present invention is
There is no particular limitation. For example, a liquid culture solution cultivated using a microorganism capable of producing ABA and a solid culture extraction aqueous solution obtained by hot water extraction of a solid culture are mentioned. When a liquid culture medium is used, it can be used without removing the cells. ABA concentration in the aqueous solution is 0.001-
5% is preferable. The pH of the aqueous solution is usually 1 to 5, and 2 to 4 is particularly preferable.

The organic solvent that is immiscible with water is not particularly limited, but is selected from the organic solvents that separate from water and dissolve ABA. For example, esters such as ethyl acetate and n-butyl acetate, alcohols such as n-butanol and cyclohexanol, ketones such as methyl ethyl ketone and methyl isobutyl ketone, ethers such as diethyl ether and ethyl isopropyl ether, chloroform, dichloroethane and the like. Halogenated hydrocarbons, etc., which may be used alone or in admixture of two or more. Although the amount of the solvent is not particularly limited, it is usually 0.1 to 3 times the weight of the aqueous solution containing ABA, and 0.1 to 1 time is used for carrying out the present invention particularly effectively. The contact method between the aqueous solution containing ABA and the organic layer is arbitrary, and may be a batch method or a continuous method.

Thus, ABA is transferred from the aqueous solution containing ABA to the organic solvent.

Next, in the present invention, (ii) (i) the organic layer containing ABA separated in the extraction step is brought into contact with an adsorbent and subjected to an adsorption step of adsorbing ABA from the organic layer to the adsorbent.

The adsorbent is not particularly limited, but is, for example, a general-purpose adsorbent such as silica gel, activated clay, synthetic adsorbent high porous polymer, or ion exchange such as gel type anion exchange resin or porous type anion exchange resin. Resin can be used. An organic layer containing ABA is brought into contact with these adsorbents. The contact method is arbitrary and may be a batch method or a continuous method.

Thus, ABA is adsorbed on the adsorbent from the organic layer.

Further, in the present invention, the remaining organic layer having the ABA adsorbed in the (iii) (ii) adsorption step is subjected to (i) the step of recycling to the extraction step.

The remaining organic layer having ABA adsorbed in the (ii) adsorption step is recycled to the above-mentioned (i) extraction step and can be used as it is.

On the other hand, in the present invention, (iv) (ii) the step of isolating the ABA from the adsorbent adsorbing the ABA separated in the adsorption step is provided. The method for isolating ABA from the adsorbent containing ABA separated in the adsorption step is not particularly limited. For example, if it is adsorbed on a general-purpose adsorbent,
After eluting with an organic solvent capable of eluting A, acetone or the like, the solvent may be concentrated and crystallized. When an anion exchange resin is used, a method of passing an acidic eluent such as a hydrochloric acid-acetone solution to elute ABA and then concentrating and crystallizing the solvent can be mentioned.

The ABA thus precipitated can be isolated by filtration or centrifugation. The isolated ABA is obtained by a known crystallization and purification method, that is, after making a uniform solution with an organic solvent, the uniform solution is cooled or concentrated to give ABA.
Can be crystallized to give a highly pure product.

(I) The extraction tank in the extraction step is not particularly limited, but a general extraction tank and an extraction tower or a culture tank are used. The agitation of the extraction tank is 100 ~ depending on the type of the tank.
A rotation speed of 1,000 rpm is preferred. The temperature of the extraction tank is not particularly limited, but it is preferably controlled at 0 to 50 ° C.

An embodiment of the present invention will be described below with reference to the flow chart of FIG.

In the extraction step (1) of the extraction step, the aqueous solution containing ABA is contacted with an organic solvent immiscible with water. Using the pump of (2), the organic layer containing ABA in the extraction tank of (1) is supplied to the packed column of (3) containing the adsorbent in the adsorption step, and the adsorbent and ABA
The organic layer containing is contacted. The ABA-adsorbed organic layer in the packed tower of (3) is circulated in the extraction tank of (1) of the extraction step.
The eluate in the eluent tank is supplied to the packed column in (3) by the pump in (5), and the eluate containing ABA is fed to the isolation step to isolate ABA.

[0022]

EXAMPLES The present invention will be illustrated concretely by the following examples.

Example 1 Glucose 4%, yeast extract 0.3%, potassium 1 phosphate 0.2%, potassium chloride 0.1%, magnesium sulfate hydrate 0.02%, polyoxyethylene sorbitan monostearate 0 Liquid medium consisting of 0.01% was added to 5 500 ml baffled flasks, 50 ml at a time, at 120 ° C.
It was sterilized by autoclaving for 5 minutes. Botrytis Cinerea F
4% of the pre-cultured bacterial solution of ERM P-6156 was inoculated,
Cultivated at a rotation speed of 180 rpm and an amplitude of 7 cm at 25 ° C for 7 days,
A culture solution having an ABA concentration of 0.05% was obtained.

In a 3 liter jar fermenter, 1,000 g of this culture solution and 500 of methyl isobutyl ketone solvent.
ml and acetone 150 ml, 300 rpm at room temperature
It was stirred at. AB of the supernatant of this jar fermenter
A layer of methyl isobutyl ketone / acetone extracted from A
A polystyrene pumping resin, Amberlite XAD-2, was passed through a column filled with 250 ml of a metering pump at a rate of 5 ml / min, and A in the methylbutylketone / acetone layer was passed through the column.
BA was adsorbed. The methyl isobutyl ketone / acetone layer on which ABA was adsorbed was recycled to a 3 liter jar fermenter containing the culture solution.

After this operation was carried out for 7 hours, 500 ml of 20 Amberlite XAD-2 resin on which ABA was adsorbed was used.
% Acetone water was passed through the column to elute ABA. The eluate was concentrated under reduced pressure and then dried under reduced pressure to obtain 0.43 g of ABA having a purity of 76.4%.

Example 2 In place of the polystyrene-based porous adsorption resin Amberlite XAD-2 of Example 1, 250 ml of a strongly basic anion exchange resin Amberlite IRA-400AA (OH) was used, and in place of 20% acetone water. To 1N-Hcl / 20%
The same procedure as in Example 1 was repeated except that acetone water was used to obtain 0.45 g of ABA having a purity of 77.5%.

Comparative Example 1 1,000 g of the same culture solution as in Example 1 and 500 ml of methyl isobutyl ketone solvent were placed in a 3 liter jar fermenter and stirred at room temperature at 300 rpm for 1 hour. Only the ketone layer was extracted. Further, 500 ml of methyl isobutyl ketone solvent was charged and the same operation was repeated. This operation was performed 3 times in total, and the obtained methyl isobutyl ketone layer was heated in a warm water bath at about 45 ° C. for 2 times.
Concentrated under a reduced pressure of 5 torr to obtain 60.5% pure ABA0.
78 g are obtained.

[0028]

According to the present invention, the amount of the organic solvent used is small, and the organic layer extracted with the organic solvent is not concentrated but is efficiently and selectively produced in a high yield from the aqueous solution containing ABA. ABA can be isolated. In addition, the isolated ABA
Reduces the subsequent refining load and is industrially inexpensive high-purity ABA
It is possible to obtain

[Brief description of drawings]

FIG. 1 is a flow chart showing an embodiment of the present invention.

[Explanation of symbols]

 (1) Extraction tank (2) Pump (3) Packed tower (4) Eluent tank (5) Pump

Claims (2)

[Claims] 1. (i) an extraction step of bringing an aqueous solution containing abscisic acid into contact with an organic solvent immiscible with water to transfer the abscisic acid from the aqueous solution to the organic solvent,
(Ii) the organic layer containing the abscisic acid separated in the (i) extraction step, contact with an adsorbent, adsorption step for adsorbing the abscisic acid from the organic layer to the adsorbent,
(iii) the step of recycling the remaining organic layer in which the abscisic acid is adsorbed in the (ii) adsorption step to the (i) extraction step and (iv) the abscisic acid adsorbed in the (ii) adsorption step A method for isolating abscisic acid, comprising the step of isolating the abscisic acid from an adsorbent. 2. The method for isolating abscisic acid according to claim 1, wherein the contacting and recycling of steps (i) to (iii) are carried out continuously.